CN113663089B - Ionizable lipid nanoparticle composition, preparation method and application - Google Patents

Ionizable lipid nanoparticle composition, preparation method and application Download PDF

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CN113663089B
CN113663089B CN202110713233.6A CN202110713233A CN113663089B CN 113663089 B CN113663089 B CN 113663089B CN 202110713233 A CN202110713233 A CN 202110713233A CN 113663089 B CN113663089 B CN 113663089B
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lipid
lipid nanoparticle
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CN113663089A (en
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黄渊余
郭帅
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Beijing Institute of Technology BIT
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Abstract

The invention relates to lipid nanoparticles and discloses an ionizable lipid nanoparticle composition comprising a key lipid iBL0104, a helper lipid selected from one of DSPC, DPPC, POPC, DOPE and DEPC, a co-lipid, cholesterol, a PEG lipid, a metal chelate and a therapeutic drug. The invention also discloses a preparation method of the ionizable lipid nanoparticle composition and application of the ionizable lipid nanoparticle composition in tumor diagnosis and treatment.

Description

Ionizable lipid nanoparticle composition, preparation method and application
Technical Field
The invention relates to lipid nanoparticles, and in particular relates to an ionizable lipid nanoparticle composition. The invention also relates to a preparation method of the ionizable lipid nanoparticle composition and application of the ionizable lipid nanoparticle composition.
Background
The search for small interfering RNA (siRNA) as a therapeutic modality has increased dramatically over the past decade. Currently, 4 siRNA therapies have been approved for clinical use. siRNA is a double-stranded RNA of 20 to 25 nucleotides in length, and has many different biological uses. It is known that siRNA is mainly involved in the phenomenon of RNA interference (RNAi) and regulates the expression of genes in a specific manner. In addition, they are involved in some response pathways related to RNAi, such as antiviral mechanisms or changes in chromatin structure.
siRNA usually needs to enter into the cell to be able to exert its function of regulating gene expression well, and thus it is necessary to establish an effective and clinically applicable in vivo administration system to mediate efficient entry and rapid endosomal escape of siRNA. The lipid nano-carrier has the advantages of controllable preparation, high encapsulation efficiency, high transport efficiency, good biocompatibility and the like, and is widely applied to nucleic acid delivery and clinical research. Patisiran, the first commercial siRNA therapeutic in the world, was delivered via a lipid formulation with Dlin-MC3-DMA as the critical lipid. Lipid nanoparticles have been the primary vehicle for siRNA entry into the cell interior to date. Ionizable lipid nanoparticles are lipid nanoparticles that are neutral in blood and are ionized and cleaved in the acidic environment inside cells to release the carried drug, and it has been reported that, when dissociated lipids stay in the acidic endosome, they are positively charged and interact with anionic lipids such as phosphatidylserine on the endosome membrane, resulting in nuclear endosome membrane disruption, efficient nucleic acid escape and cytosolic release. Thus, the toxicity in vivo is lower, and the drug can be effectively carried into the cell.
The tumor is a new organism formed by that certain cells of local tissues lose normal regulation and control on the growth of the local tissues at the gene level under the action of various carcinogenic factors, so that the local tissues are clonally abnormally proliferated. Tumors are generally classified into two major groups, benign and malignant. Malignant tumors grow rapidly, are easy to generate early metastasis, relapse and have poor prognosis, and need early treatment. Thus, visualization of pathogenic sites such as tumor tissue is critical for clinical treatment, which facilitates early diagnosis, drug tracking, and observation of disease progression and metastasis. Magnetic Resonance Imaging (MRI) has high spatial resolution and deep tissue penetration ability without radiation, can visualize the three-dimensional structure of tissue, and is commonly used for imaging and early diagnosis of diseases such as tumor. However, MRI is less sensitive than Positron Emission Tomography (PET) and optical imaging, resulting in poor contrast resolution in distinguishing between tumors and adjacent normal tissue. Usually, a metal chelate needs to be injected as a developer, and the metal chelate is gathered at pathogenic sites such as tumor tissues and the like, so that the pathogenic sites such as the tumor tissues and the like are better imaged, and visualization is realized.
At present, paramagnetic gadolinium ion chelates, such as Gd-DTPA, gd-DOTA and Gd-HPDO3A, are used clinically primarily to enable visualization of tumor tissue, which can increase contrast between MRI regions by shortening the longitudinal and transverse relaxation times of the surrounding water protons. The metal ion chelate can also be carried into cells of tumor tissues through the lipid nanoparticles, so that the visualization of the tumor tissues is realized. The existing lipid nanoparticles are difficult to simultaneously and reliably carry metal chelates and therapeutic drugs, balance is kept between visualization and treatment, not only is the diagnosis effect unsatisfactory, but also the treatment effect is influenced. Therefore, there is a pressing need in the art for a nanoparticle composition that is easy to prepare, has good biocompatibility, and can achieve simultaneous diagnosis and treatment.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an ionizable lipid nanoparticle composition which can realize diagnosis and treatment functions simultaneously and has good biocompatibility.
A further object of the present invention is to provide a method for preparing an ionizable lipid nanoparticle composition, which can conveniently prepare an ionizable lipid nanoparticle composition that can simultaneously perform diagnostic and therapeutic functions.
The technical problem to be solved by the invention is to provide an application of the ionizable lipid nanoparticle composition in tumor diagnosis and treatment.
To achieve the above objects, the present invention provides in a first aspect an ionizable lipid nanoparticle composition comprising a key lipid of iBL0104, a helper lipid selected from one of DSPC, DPPC, POPC, DOPE and DEPC, cholesterol, a PEG lipid, a metal chelate and a therapeutic drug.
The ionizable lipid nanoparticle composition according to the invention, said PEG lipid being selected from the group consisting of DSPE-PEG 2000 、DMG-PEG 2000 、DPPE-PEG 2000 And DMA-PEG 2000 One of them.
The ionizable lipid nanoparticle composition according to the present invention, said metal chelate being selected from one of DTPA-BSA (Gd), gd-DTPA and Gd-BOPTA.
According to the ionizable lipid nanoparticle composition, the therapeutic drug is a nucleic acid molecule, and the nucleic acid molecule is selected from one or more of siRNA, mRNA, ASO, plasmid or sarRNA.
Preferably, the ionizable lipid nanoparticle composition of the present invention further comprises a targeting peptide.
Further preferably, the PEG lipid and the targeting peptide are the same species DSPE-PEG2000-RGD.
In a second aspect, the present invention provides a method for preparing the ionizable lipid nanoparticle composition provided in the first aspect, comprising the steps of: 1) Adding iBL0104, helper lipid, cholesterol, PEG lipid and metal chelate in a set proportion into anhydrous ethanol according to a total amount of 0.015-150mg/mL to obtain an ionizable lipid solution; 2) Rapidly adding the lipid solution into 1-6 times of sodium citrate buffer solution, and stirring to obtain ionizable lipid nanoparticle solution; 3) Adding a certain amount of therapeutic drug into a sodium citrate buffer solution containing ethanol, and dissolving to obtain a therapeutic drug solution of 0.0025-2.5 mg/mL; 4) Mixing the ionizable lipid nanoparticle solution and the therapeutic drug solution in a ratio of 0.5-2:1, and carrying out water bath to obtain an ionizable lipid nanoparticle solution for wrapping the therapeutic drug; 5) Dialyzing with 100kD dialysis membrane to obtain the ionizable lipid nanoparticle composition of the invention.
According to the method of the invention, the PEG lipid is DMG-PEG2000, the metal chelate is DTPA-BSA (Gd), and the therapeutic drug is siRNA; in the step 1), the proportion of iBL0104 is 20-60%, the proportion of cholesterol is 10-50%, the proportion of DTPA-BSA (Gd) is 10-50%, the proportion of DSPC is 1-20%, and the proportion of DMG-PEG2000 is 0.1-5%.
According to the method of the invention, the PEG lipid is DSPE-PEG2000-RGD, the metal chelate is DTPA-BSA (Gd), and the therapeutic drug is siRNA; in the step 1), the proportion of iBL0104 is 20-60%, the proportion of cholesterol is 10-50%, the proportion of DTPA-BSA (Gd) is 10-50%, the proportion of DSPC is 1-20%, and the proportion of DSPE-PEG2000-RGD is 0.1-5%.
In a third aspect, the present invention provides a use of the ionizable lipid nanoparticle composition provided in the first aspect of the present invention in diagnosis and treatment of tumors.
Through the technical scheme, the ionizable lipid nanoparticle composition disclosed by the invention adopts iBL0104 as key lipid to form a skeleton main body of the lipid nanoparticle, so that the lipid nanoparticle can better penetrate through a cell membrane. 5363 the structure of iBL0104 can be expressed as:
Figure BDA0003134444260000041
the core lipid also has proper pKa (pKa is 5.90), is neutral in human peripheral environment (blood and tissue fluid), has low toxicity and strong biocompatibility, can be ionized in intracellular acidic environment (endosome/lysosome), can exert excellent endosome escape effect, effectively releases therapeutic drugs and improves the therapeutic effect of the therapeutic drugs. Meanwhile, the metal chelate is used, so that metal can be chelated on the nano particles, and the nano particles enter cells, thus ensuring the MRI sensitivity of pathogenic sites and providing the visibility of the pathogenic sites. The ionizable lipid nanoparticle composition can effectively penetrate through cell membranes, improves the content of metal chelates and therapeutic drugs in cells, can simultaneously realize ideal contrast effect and effective in-vivo treatment, and really realizes imaging-guided treatment. The ionizable lipid nanoparticle composition has excellent biocompatibility and degradability, no obvious toxic or side effect and good clinical application prospect. The preparation method of the ionizable lipid nanoparticle composition can conveniently prepare the ionizable lipid nanoparticle composition, and has the advantages of low equipment requirement and reliable process.
Drawings
FIG. 1 is a MALDI-TOF-MS analysis mass spectrum of cRGD peptide, DSPE-PEG2000-MAL and DSPE-PEG2000-cRGD binding;
FIG. 2 is a graph of the effect of different doses of Gd preparations on GAP iLNPs imaging;
FIG. 3 is a graph showing the results of the measurement of the effect of the ionizable lipid nanoparticle composition of the present invention on the activity of cells;
FIG. 4 is a fluorescent quantitative assay of the relative expression of PLK1 mRNA in HepG2-Luc cells after treatment with GAP iLNPs;
FIG. 5 is a fluorescent quantitation assay of the relative expression of PLK1 mRNA in HepG2-Luc cells after treatment with GARP iLNPs;
FIG. 6 is a graph of the gene silencing effects of GAP/sipLK1 and GARRP/sipLK 1;
FIG. 7 is a graph showing the results of the protein level of PLK1 measured by Western blotting;
FIG. 8 is a graph showing the results of quantitative analysis of the level of PLK1 protein;
FIG. 9 is a graph of the pKa measurements for one embodiment of the ionizable lipid nanoparticle composition of the present invention;
FIG. 10 is a graph of fluorescence imaging of the ionizable lipid nanoparticle composition of the present invention in vivo in mice;
FIG. 11 is a graph of the distributed fluorescence images of the ionizable lipid nanoparticle composition of the present invention in mouse tumor tissues and major organs;
FIG. 12 is a graph of MRI images of a mouse liver cancer model using the ionizable lipid nanoparticle composition of the present invention;
FIG. 13 is a graph of the effect of ionizable lipid nanoparticle compositions of the present invention on tumor tissue growth;
FIG. 14 is a graph of the effect of ionizable lipid nanoparticle compositions of the present invention on tumor tissue PLK1 mRNA expression;
FIG. 15 is a graph of the effect of ionizable lipid nanoparticle compositions of the present invention on survival time in a mouse liver cancer model;
FIG. 16 is a graph of the effect of ionizable lipid nanoparticle compositions of the present invention on body weight in a mouse liver cancer model;
FIG. 17 is a graph of the effect of ionizable lipid nanoparticle compositions of the present invention on liver and kidney function in a mouse liver cancer model;
FIG. 18 is a graph showing the histopathological effects of the ionizable lipid nanoparticle composition of the present invention on the major organs of a mouse liver cancer model.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For numerical ranges, each range between its endpoints and individual point values, and each individual point value can be combined with each other to give one or more new numerical ranges, and such numerical ranges should be construed as specifically disclosed herein.
The following detailed description of the embodiments of the method for preparing norfloxacin magnetic molecularly imprinted nanoparticles according to the present invention is provided in conjunction with the accompanying drawings, and it should be understood that the embodiments described herein are only for illustrating and explaining the present invention, and the scope of the present invention is not limited to the following embodiments.
In the examples of the present invention, the mass spectrometer used was a mass spectrometer manufactured by Bruker Daltonics, usa, model MALDI-TOF-MS; the magnetic resonance imager is an animal magnetic resonance imager which is produced by German Bruk and has the model number of 7.0-T; the fluorescent real-time quantitative PCR instrument is a fluorescent quantitative instrument which is produced by thermo Fisher Scientific and has the model of qRT-PCR; the chemiluminescence imaging system comprises: bio-Rad, bossier City, LA; the living body imaging system is as follows: kodak In-Vivo Image System FXPro, care stream Health, USA; hepG2-Luc cells were purchased from ATCC cell banks; both the TRIzol reagent and the reverse transcription kit adopt Vazyme of Nanjing China; the BCA protein assay kit is produced by CWBIO, and the batch number is CW0014; mouse anti-PLK 1 monoclonal antibodies were obtained from U.S. cell signaling technologies (1, ab30394); horseradish peroxidase-conjugated antibodies were from ZSJQB, inc (1; other reagents were all commercially available.
The preparation method of the ionizable lipid nanoparticle composition, the ionizable lipid nanoparticle composition and the application thereof of the present invention are described in detail by examples below.
Example 1
This example was used to prepare ionizable lipid nanoparticle compositions without targeting function.
iBL0104, cholesterol, DTPA-BSA (Gd), DSPC, DMG-PEG2000 and siRNA (sinC, sipLK1 or Cy 5-siRNA) are used to prepare lipid nanoparticles without targeting peptide and with diagnosis and treatment functions, and the lipid nanoparticles are named GAP/siRNA iLNPs.
1) iBL0104, cholesterol, DTPA-BSA (Gd), DSPC and DMG-PEG were taken in the proportions shown in Table 1, respectively 2000 And dissolved in absolute ethyl alcohol to form 1.5mg/mL of organic phase ethanol solution.
Table 1: raw material proportioning table of GAP/siRNA iLNPs
Figure BDA0003134444260000071
Figure BDA0003134444260000081
2) The organic phase was injected at high speed into a 3-fold volume of 50mMpH4.0 sodium citrate buffer solution and stirred at 2000-6000rpm for 3 minutes to form GAP iLNPs.
3) GAP iLNP was added to 50mM sodium citrate buffer pH4.0 containing 25% ethanol, and dissolved with stirring to form a solution of 0.025 mg/mL.
4) GAP iLNP was mixed with an equal amount of siRNA and water-bathed at a temperature of 50 ℃ for 30 minutes.
5) The solution after the water bath is filled in a dialysis membrane of 100kD and is placed in 1 XPBS for dialysis for at least 2 hours to obtain the GAP/siRNA iLNPs of the invention.
In the GAP/siRNA iLNPs prepared in this example, the mass ratio of total lipid to siRNA was about 15:1.
example 2
This example was used to prepare ionizable lipid nanoparticle compositions without targeting function.
1) The proportion of iBL0104, cholesterol, DTPA-BSA (Gd), DPPC and DPPE-PEG were taken as 20 2000 And dissolved in absolute ethyl alcohol to form 0.015mg/mL of organic phase ethanol solution.
2) The organic phase was injected at high speed into an equal volume of 50mM pH4.0 sodium citrate buffer solution and stirred at 2000-6000rpm for 3 minutes to form GAP iLNPs.
3) GAP iLNP was added to 50mM sodium citrate buffer pH4.0 containing 25% ethanol and dissolved with stirring to form a 0.0025mg/mL solution.
4) GAP iLNP was mixed with mRNA in a volume ratio of 1:2 and water-bathed at 50 ℃ for 30 minutes.
5) The solution after the water bath is filled in a dialysis membrane of 100kD and is dialyzed in 1 XPBS for at least 2 hours to obtain the GAP/mRNA iLNPs of the invention.
Example 3
This example was used to prepare ionizable lipid nanoparticle compositions without targeting function.
1) Taking iBL0104, cholesterol, gd-DTPA, DOPC and DSPE-PEG according to the proportion of 50 2000 And dissolving in absolute ethyl alcohol to form 150mg/mL of organic phase ethanol solution.
2) The organic phase was injected at high speed into a 6-fold volume of 50mM pH4.0 sodium citrate buffer solution and stirred at 2000-6000rpm for 3 minutes to form GAP iLNPs.
3) GAP iLNP was added to 50mM sodium citrate buffer pH4.0 containing 25% ethanol, and dissolved with stirring to form a 2.5mg/mL solution.
4) GAP iLNP was mixed with ASO at a volume ratio of 2:1 and bathed at 50 ℃ for 30 minutes.
5) The solution after the water bath is filled in a dialysis membrane of 100kD and dialyzed in 1 XPBS for at least 2 hours to obtain the GAP/ASO iLNPs of the invention.
Example 4
This example was used to prepare ionizable lipid nanoparticle compositions without targeting function.
1) Taking iBL0104, cholesterol, gd-BOPTA, DOPC and DMA-PEG according to the proportion of 50 2000 And dissolving in absolute ethyl alcohol to form 150mg/mL of organic phase ethanol solution.
2) The organic phase was injected at high speed into a 6-fold volume of 50mM pH4.0 sodium citrate buffer solution and stirred at 2000-6000rpm for 3 minutes to form GAP iLNPs.
3) GAP iLNP was added to 50mM sodium citrate buffer pH4.0 containing 25% ethanol and dissolved with stirring to form a 2.5mg/mL solution.
4) GAP iLNP was mixed with plasmid 2:1 by volume and bathed at 50 ℃ for 30 minutes.
5) The solution after the water bath is filled in a dialysis membrane of 100kD and is dialyzed in 1 XPBS for at least 2 hours to obtain the GAP/plasmid iLNPs of the invention.
Example 5
This example was used to prepare ionizable lipid nanoparticle compositions with targeting functionality.
iBL0104, cholesterol, DTPA-BSA (Gd), DSPC, DSPE-PEG2000-cRGD and siRNA (sinC, sipLK1 or Cy 5-siRNA) are used for preparing lipid nanoparticles with targeting peptide c (GRGDSPKC) and with diagnosis and treatment functions, and the lipid nanoparticles are named as GARP/siRNA iLNPs.
1) Synthesis of DSPE-PEG 2000 -cRGD. The cRGD peptide and the DSPE-PEG2000-MAL are mixed according to the mass ratio of 1:5 in 100mM HEPES buffer solution (pH7.0), at 4 ℃ after stirring for 48 hours, in 2kDa dialysis bag, in deionized water dialysis for 24 hours.
The resulting solution was identified using a mass spectrometer, and the results are shown in FIG. 1, demonstrating that the DSPE-PEG2000-cRGD to be synthesized was obtained.
2) iBL0104, cholesterol, DTPA-BSA (Gd), DSPC and DSPE-PEG were taken in the proportions shown in Table 2, respectively 2000 -cRGD, dissolved in absolute ethanol to form 1.5mg/mL of organic phase ethanol solution.
Table 2: GARP/siRNAilNPS raw material proportioning table
Figure BDA0003134444260000101
3) The organic phase was injected at high speed into a 3-fold volume of 50mM pH4.0 sodium citrate buffer solution and stirred at 2000-6000rpm for 3 minutes to form GARP iLNPs.
4) GARP iLNP was added to 50mM sodium citrate buffer pH4.0 containing 25% ethanol and dissolved with stirring to form a 0.025mg/mL solution.
5) GARP iLNP was mixed with an equal amount of siRNA and bathed at 50 ℃ for 30 minutes.
6) Solutions after the water bath all formulations were dialyzed in dialysis membrane of 1 × PBS for at least 2 hours to obtain GARP/siRNA iLNPs of the invention.
The GARP/siRNA iLNPs prepared in this example had a mass ratio of total lipid to siRNA of about 15:1.
example 6
This example was used to prepare ionizable lipid nanoparticle compositions with targeting functionality.
1) Synthesis of DSPE-PEG 2000 -cRGD. The cRGD peptide and the DSPE-PEG2000-MAL are mixed according to the mass ratio of 1:5 was dissolved in 100mM HEPES buffer (pH 7.0), stirred at 4 ℃ for 48 hours, packed in a 2kDa dialysis bag, and dialyzed in deionized water for 24 hours.
2) Taking iBL0104, cholesterol, DTPA-BSA (Gd), DOPE and DSPE-PEG according to the proportion of 20 2000 -cRGD, dissolved in absolute ethanol to form a 0.015mg/mL ethanol solution of the organic phase.
3) The organic phase was injected at high speed into an equal volume of 50mM pH4.0 sodium citrate buffer solution and stirred at 2000-6000rpm for 3 minutes to form GARP iLNPs.
4) GARP iLNP was added to 50mM pH4.0 sodium citrate buffer solution containing 25% ethanol and dissolved with stirring to form a 0.0025mg/mL solution.
5) GARP iLNP was mixed with siRNA in a volume ratio of 1:2 and water bath was carried out at a temperature of 50 ℃ for 30 minutes.
6) The solution after the water bath is filled in a dialysis membrane of 100kD and dialyzed in 1 XPBS for at least 2 hours to obtain the GARP/siRNAiLNPs of the invention.
Example 7
This example was used to prepare ionizable lipid nanoparticle compositions with targeting functionality.
1) Synthesis of DSPE-PEG 2000 -cRGD. Mixing cRGD peptide and DSPE-PEG2000-MALThe mass ratio is 1:5 in 100mM HEPES buffer (pH 7.0), stirred at 4 ℃ for 48 hours, packed in a 2kDa dialysis bag, and dialyzed in deionized water for 24 hours.
2) iBL0104, cholesterol, DTPA-BSA (Gd), DEPC and DMG-PEG were taken at the ratio of 50 2000 And dissolving in absolute ethyl alcohol to form 150mg/mL of organic phase ethanol solution.
3) The organic phase was injected at high speed into a 6-fold volume of 50mM pH4.0 sodium citrate buffer solution and stirred at 2000-6000rpm for 3 minutes to form GARP iLNPs.
4) GARP iLNP was added to 50mM sodium citrate buffer pH4.0 containing 25% ethanol and dissolved with stirring to form a 2.5mg/mL solution.
5) GARP iLNP was mixed with saRNA in a volume ratio of 2:1 and bathed at 50 ℃ for 30 minutes.
6) The solution after the water bath is filled in a dialysis membrane of 100kD and is placed in 1 XPBS for dialysis for at least 2 hours to obtain the GARP/sarnaiLNPs of the invention.
Examples of the experiments
(1) Extra-body magnetic resonance imaging
To evaluate the contrast agent effect of GARP iLNPs, the GAP/siRNA iLNPs solution prepared in example 1 was subjected to magnetic resonance imaging using an animal magnetic resonance imager with a repetition Time (TR) =3000ms and an echo Time (TE) =40ms. As shown in FIG. 2, the T1 value of GAP iLNPs decreased and the MRI contrast effect increased with the increase of DTPA-BSA (Gd) content. GAP32, GAP35 and GAP60 all exhibit the desired contrast effect compared to PBS.
(2) Cytotoxicity assays
MTT assay was used to detect the cytotoxic effects of GARP iLNPs on cells. When HepG2-Luc cells were in the logarithmic growth phase, 1 ten thousand cells were seeded into a 96-well plate at 100. Mu.L of cell suspension per well and incubated for 24 hours. GARP35iLNPs from example 5 and GAP35 iLNPs from example 1 were then used as controls, and untreated cells were used as negative controls. GARP35iLNPs and GAP35 iLNPs were transfected separately in fresh whole DMEM containing 10% fetal bovine serum for 24 hours. After 95. Mu.L of fresh DMEM and 5. Mu.L of MTT (5 mg/mL) were added to each well, and incubated at 37 ℃ for 4 hours, the absorbance was measured at 540nm with a microplate reader after the formazan crystals were completely dissolved, and the cell viability was calculated using 650nm as a reference wavelength. Specifically, MTT method was used to detect the cytotoxic effects of GARP35iLNPs and GAP35 iLNPs on HepG2-Luc cells. As shown in fig. 3, all lipid nanoparticle formulations were not significantly cytotoxic even when siRNA transfection concentrations reached 600 nM.
(3) Fluorescent real-time quantitative PCR
Fluorescent real-time quantitative PCR (qRT-PCR) detects gene silencing efficiency. HepG2-Luc cells were seeded in 6-well plates at a cell density of 2X 105 cells/well. After 24 hours, GARP35/sipLK1 lipid nanoparticles and GAP35/sipLK1 lipid nanoparticles were transfected into cells at final siRNA concentrations of 50nM and 150nM for 4 hours. Total RNA extraction and reverse transcription were performed using TRIzol reagent and reverse transcription kit, respectively, according to standard manufacturing instructions. Quantitative determination of cDNA was carried out by qRT-PCR system using SYBR Green PCR Master Mix as template. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene was used as an internal control.
The gene inhibition efficiency of GAP/siRNAILNPs at different lipid component molar ratios was analyzed against siRNA of PLK1 (siPLK 1) using a fluorescent real-time quantitative PCR instrument. As shown in fig. 4, GAP35/siPLK1iLNP showed the highest gene silencing activity compared to other iLNP preparations. Furthermore, as shown in FIG. 5, GARP/sipLK 1iLNP showed better gene silencing activity compared to other GAP/sipLK1 iLNP. GAP35 and GARP35iLNPs are generally superior to other nanoparticles in terms of magnetic resonance imaging and gene silencing effects.
(4) Western blot
The expression of the PLK1 protein of the HepG2-Luc cells is detected by western blotting. Cells were treated in the same way as real-time fluorescent PCR detection. Whole cell proteins were extracted with 1-fold passive lysis buffer containing protease inhibitor (10000X). Protein concentration was determined using BCA protein assay kit. 60. Mu.g of protein was separated by SDS-PAGE and blotted onto Nitrocellulose (NC). Blocking with 5% Bovine Serum Albumin (BSA) buffer at room temperature for 1 hour, incubating with mouse anti-PLK 1 monoclonal antibody overnight at 4 ℃, and incubating with horseradish peroxidase-conjugated antibody at room temperature for 1 hour. Spots were recorded and analyzed using a chemiluminescent imaging system.
The GARP35/sipLK 1iLNP and GAP35/sipLK1 iLNPs were further analyzed for gene silencing activity. The data show that GAP35/sipLK1 and GARP35/sipLK1 iLNPS exhibit significant gene silencing effects in vitro. GAP35/sipLK1 treated cells achieved 89.30% and 94.49% silencing efficiency at siRNA concentrations of 50nM and 150nM, respectively, and GARP35/sipLK1 treated cells achieved 96.64% and 98.74% silencing efficiency at siRNA concentrations of 50nM and 150nM, respectively. As shown in FIG. 6, the inhibition of PLK1 mRNA expression by Lipo 2000-transfected sipLK1 was 85.30%. The GARP35/sipLK 1-mediated downregulation of mRNA expression levels was significantly higher than that of GAP35/sipLK 1-mediated downregulation, suggesting that cRGD modification significantly enhances nanoparticle endocytosis in cells through RGD/integrin interaction mechanisms. As shown in fig. 7 and 8, western blot results for detecting protein expression showed the same tendency.
(5) PKa assay GARP iLNPs
To characterize the ionization performance of the GARP nanoparticles, 1mL of GARP nanoparticles (total lipid concentration: 2. Mu.M) and 1mL of nanoparticles without DTPA-BSA (Gd) (iLNPW/OGd) were prepared (total lipid concentration: 6. Mu.M). First, 100mM HEPES buffer, 100mM MES, 100mM ammonium acetate, 1300mM NaCl, and 100. Mu.M TNS were prepared as stock solutions in distilled water. A series of nanosolutions at different pH conditions were then prepared. These solutions contained 10mM HEPES, 10mM MES, 10mM ammonium acetate, 130mM NaCl, 80. Mu.M GARP NPs. Subsequently, 99. Mu.L of the nanoparticle solution and 1. Mu.L of the TNS stock solution were added to a 96-well black opaque plate, and the fluorescence values were measured at an excitation wavelength of 321nm and an emission wavelength of 445 nm. Finally, the pKa of the GARP NPs was calculated by fitting the Henderson-Hasselbach equation (GraphPad Prism v.8).
pKa values of GARP iLNPs were determined by titration with 2- (p-tolyl) -6-naphthalenesulfonic acid (TNS). As shown in fig. 9, their pKa values were observed to be 6.07 and 5.90, respectively. The pKa value of the lipid-like nanoparticle must reach or exceed 5.5, otherwise the nanoparticle can hardly exhibit ideal siRNA transport efficiency in vivo. Therefore, the prepared GARP iLNPs may trigger effective endosome escape through a membrane destabilization mechanism.
(6) In vivo tumor targeting validation in animals
Targeted accumulation of GARP iLNPs in vivo: to evaluate targeting of the GARP nanoparticles in mice, hepG2-Luc cells (5X 106 cells) were suspended in 1 XPBS (100. Mu.L), injected subcutaneously into the right axilla of female BALB/c nude mice weighing about 20g, and when the tumors grew to about 1000mm 3 At this time, 4 groups were randomly selected and injected with 1 XPBS, naked Cy5-siRNA, GAP35/Cy5-siRNA, GARP35/Cy5-siRNA, respectively. The tail vein of the tested mice is injected with siRNA, and the dosage is 1mg/kg. The mouse Cy5 fluorescence signal was detected with a living body imaging system at 1 hour, 3 hours, 6 hours, 10 hours, and 24 hours after injection, respectively. Animals in each group were sacrificed at 6, 10 and 24 hours post-injection, respectively, and then observed for tumor and major organ fluorescence signals.
The results show that the distribution of Cy5 fluorescence signals detected at different time points after injection is shown in fig. 10 for both tumor and whole body. After imaging the whole body for 6 hours, 10 hours, 24 hours, tumor tissues and major organs were isolated and imaged ex vivo, and as a result, as shown in fig. 11, the fluorescence signal at the tumor was hardly visible in the Naked Cy5-siRNA group at the time of injection for 10 hours. However, fluorescent signals were detectable at tumors injected with GAP35/siRNA and GARP 35/siRNA. At 10 and 24 hours post-injection, the fluorescence intensity of GARP35/siRNA injected mice at tumors was stronger than GAP35/siRNA injected tumors, indicating that GARP35/siRNA iLNPs are metabolized more slowly in mice than GAP35/siRNA iLNPs. The GARP35/siRNA injected mice were endocytosed with more nanoparticles due to the cRGD targeting moiety than the GAP35/siRNA injected mice. In summary, GARP iLNPs with cRGD targeted modification showed more enrichment than GAP iLNPs without cRGD modification in tumor-bearing mice.
(7) Anticancer effect of ionizable lipid formulations in Patient Derived Xenograft (PDX) model
To establish a Patient Derived Xenograft (PDX) tumor model, tumor tissue from a liver cancer patient was washed 3 times with phosphate buffer, cut into small pieces, and then transplanted to the right axilla of a mouse. All manipulations were in accordance with medical ethics and experimental animal ethics principles. Tumor growth to 100-200mm 3 When mice were randomly divided into 6 groups, (1) PBS, (2) sorafenib, (3) GAP35/sipLK1 (i.v.), (4) GARP35/sipLK1 (i.v.), (5) GAP35/sipLK1 (i.t.) and (6) GARP35/sipLK1 (i.t.). Group 3 intravenous GAP35/sipLK1, group 5 intratumoral GAP35/sipLK1, group 4 intravenous GARP35/sipLK1, group 6 intratumoral GARP35/sipLK1. The siRNA dose was 1mg/kg. The oral dose of sorafenib 30mg/kg was examined by MRI before and after administration, respectively. Here, animals were anesthetized with isoflurane before and after the first dose and fixed on MRI equipment for MRI imaging to trace tumor location and drug distribution. T1-weighted images were taken before dosing, 1 hour, 5 hours, 10 hours, and 24 hours after dosing for each group of mice. Body weight, tumor volume and animal survival were also recorded throughout the treatment. At the end of the experiment, tumor tissues are separated, and serum biochemical indexes and pathological changes of main organs and tumors are detected.
Six groups of mice were identified as G1: PBS, G2: sorafenib, G3: GAP35/sipLK1 (i.v.), G4: GARP35/sipLK1 (i.v.), G5: GAP35/sipLK1 (i.t.), and G6: GARP35/sipLK1 (i.t.), "i.v." and "i.t" represent intravenous and intratumoral injections, respectively. The results of MRI imaging of mice in the G3, G4, G5, and G6 groups before and after the first administration are shown in fig. 12 (white circles indicate tumor sites). For mice injected intravenously with GAP35/sipLK1 and GARP35/sipLK1, they showed enhanced MRI signals at the tumor tissue margins 1 hour after injection. The intratumoral injection group showed enhanced MRI signals at the injection site. Over time, the lipid complex gradually spreads in the tumor tissue. And the diffusion rate was faster for the GARP35/sipLK1 group than for the GAP35/sipLK1 group. In addition, the accumulation level of GARP35/siRNA in the tumor site in the tail vein injection group and the tumor injection group is higher than that in the GAP35/siRNA group. These results indicate that cRGD coupling on lipid nanoparticles enhances tumor targeting of the nanoformulation in vivo. And the GAP35/sipLK1 and GARP35/siRNA nano-complexes have ideal MRI tumor imaging effect.
In tumor suppression experiments, drugs were given every two days and tumor volumes were recorded daily until the end of the experiment. Such asAs can be seen in FIG. 13, the tumors in the PBS group grew rapidly, and the mean tumor volume reached more than 2000mm3 on day 5. In contrast, mice treated with GARP35/sipLK1, whether tail vein injection or intratumoral injection, had much slower tumor growth. In addition, the tumor inhibiting effect of the intratumoral injection group is generally better than that of the intravenous injection group, and the tumor growth speed of the mice of the GARP35/sipLK1 injection group is slower than that of the GAP35/sipLK1 injection group. When the mean tumor volume in PBS group reached about 2000mm 3 At time, 3 animals per group were randomly selected for sacrifice. The expression of PLK1 in tumor tissues was detected by qRT-PCR method on isolated tumors. As shown in FIG. 14, expression of PLK1 mRNA was inhibited in all groups receiving siPLK1 treatment. In addition, mice of different groups were analyzed for survival, as shown in fig. 15, and survival time was significantly prolonged in the G3, G4, G5 and G6 groups compared to the PBS group. Wherein the median survival time of the PBS-treated mice was 5 days, and the median survival time of the sorafenib-treated mice was slightly increased to 6 days. The median survival times for GAP35/sipLK1 (i.v.) and GAP35/sipLK1 (i.t.) were 11 days. GARP35/siPLK1 iLNPs (i.v.) and GARP35/PLK1 iLNPs (i.t.) extended median survival of mice to 13 days and 14 days, respectively.
The body weight of the mice was continuously measured throughout the treatment period of the experiment, and as shown in fig. 16, the body weight of the mice did not change significantly. It is well known that spleen weight of immunodeficient BALB/c nude mice is significantly higher than that of normal mice. Thus, assuming an improvement in health status after GARP/sipLK1 treatment, spleen weight loss may result. At the end of the experiment, blood samples and major organs of mice were subjected to serum biochemical analysis and histopathological examination, respectively. As shown in fig. 17 and 18, the biomarkers of liver and kidney function of each group of animals were not significantly changed, and major organs were not significantly changed in histopathology.
In conclusion, the ionizable lipid nanoparticle composition disclosed by the invention can be used for efficiently delivering a therapeutic agent and realizing efficient drug release. The nanoparticle composition with the targeting peptide can reach a focus part more in a targeting manner, and simultaneously realizes a high-efficiency contrast effect and a high-efficiency treatment effect, and has high biological safety. The ionizable lipid nanoparticle composition is doped with the metal chelate with an image, so that lesions such as tumor tissues and the like can be better imaged. The nanoparticle composition provided by the invention has strong functions and can synchronously realize image-guided treatment. The method for preparing the ionizable lipid nanoparticle composition can conveniently prepare the ionizable lipid nanoparticle composition which simultaneously realizes diagnosis and treatment functions. The ionizable lipid nanoparticle composition disclosed by the invention can realize image guidance and delivery of tumor-targeted drugs/siRNA, can be applied to tumor diagnosis and treatment, can play a role in diagnosis and treatment at the same time, and has a good clinical application prospect.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including various technical features being combined in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims (8)

1. An ionizable lipid nanoparticle composition comprising a key lipid iBL0104, a helper lipid selected from one of DSPC, DPPC, POPC, DOPE and DEPC, cholesterol, PEG lipid, metal chelate and therapeutic drug;
the iBL0104 has a structural formula as follows:
Figure FDA0003724360170000011
2. the ionizable lipid nanoparticle composition of claim 1, wherein said PEG lipid is selected from DSPE-PEG 2000 、DMG-PEG 2000 、DPPE-PEG 2000 And DMA-PEG 2000 One of them.
3. The ionizable lipid nanoparticle composition of claim 1, wherein said metal chelate is selected from one of Gd-DTPA-BSA, gd-DTPA and Gd-BOPTA.
4. The ionizable lipid nanoparticle composition of claim 1, wherein said therapeutic agent is a nucleic acid molecule selected from one or more of siRNA, mRNA, ASO, plasmid, or saRNA.
5. A method for preparing the ionizable lipid nanoparticle composition of any one of claims 1-4, comprising the steps of:
1) Adding iBL0104, helper lipid, cholesterol, PEG lipid and metal chelate in a set proportion into absolute ethanol according to a total amount of 0.015-150mg/mL to obtain ionizable lipid solution;
2) Rapidly adding the lipid solution into 1-6 times of sodium citrate buffer solution, and stirring to obtain ionizable lipid nanoparticle solution;
3) Adding a certain amount of therapeutic drug into a sodium citrate buffer solution containing ethanol, and dissolving to obtain a therapeutic drug solution of 0.0025-2.5 mg/mL;
4) Mixing the ionizable lipid nanoparticle solution and the therapeutic drug solution in a ratio of 0.5-2:1, and carrying out water bath to obtain an ionizable lipid nanoparticle solution for wrapping the therapeutic drug;
5) Dialyzing with 100kD dialysis membrane to obtain ionizable lipid nanoparticle composition.
6. The method of claim 5, wherein the PEG lipid is DMG-PEG 2000 The metal chelate is Gd-DTPA-BSA, and the therapeutic drug is siRNA; in the step 1), the proportion of iBL0104 is 20-61%, the proportion of cholesterol is 10-60%, the proportion of Gd-DTPA-BSA is 10-60%, the proportion of DSPC is 1-20%, and the DMG-PEG 2000 The proportion of (B) is 0.1-5%.
7. According to the claimsThe method according to claim 5, wherein the PEG lipid is DSPE-PEG 2000 -RGD, said metal chelate being Gd-DTPA-BSA, said therapeutic drug being siRNA; in the step 1), the proportion of iBL0104 is 20-61%, the proportion of cholesterol is 10-60%, the proportion of Gd-DTPA-BSA is 10-60%, the proportion of DSPC is 1-20%, and the DSPE-PEG 2000 The proportion of RGD is 0.1-5%.
8. Use of the ionizable lipid nanoparticle composition of any one of claims 1-4 for the preparation of a tumor diagnostic and a tumor therapeutic drug.
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